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Abstract:

The present invention relates to glucoamylase variants having reduced
sensitivity to protease nicking. The present invention also relates to
polynucleotides encoding the variants; nucleic acid constructs, vectors,
and host cells comprising the polynucleotides; and methods of using the
variants.

Claims:

1. A glucoamylase variant, comprising a substitution at least at a
position corresponding to positions 79 of the mature polypeptide of SEQ
ID NO: 2, wherein the variant has glucoamylase activity.

2. The glucoamylase variant of claim 1, selected from the group
consisting of: a) a polypeptide having at least 65% sequence identity to
the mature polypeptide of SEQ ID NO: 2; b) a polypeptide encoded by a
polynucleotide that hybridizes under low stringency conditions with (i)
the mature polypeptide coding sequence of SEQ ID NO: 1, or (ii) the
full-length complement of (i); c) a polypeptide encoded by a
polynucleotide having at least 65% identity to the mature polypeptide
coding sequence of SEQ ID NO: 1; and d) a fragment of the mature
polypeptide of SEQ ID NO: 2, which has glucoamylase activity.

3. The glucoamylase variant of claim 1 or 2, which is a variant of a
parent glucoamylase selected from the group consisting of: a) a
polypeptide having at least 65% sequence identity to the mature
polypeptide of SEQ ID NO: 2; b) a polypeptide encoded by a polynucleotide
that hybridizes under low stringency conditions with (i) the mature
polypeptide coding sequence of SEQ ID NO: 1, or (ii) the full-length
complement of (i); c) a polypeptide encoded by a polynucleotide having at
least 65% identity to the mature polypeptide coding sequence of SEQ ID
NO: 1; and d) a fragment of the mature polypeptide of SEQ ID NO: 2, which
has glucoamylase activity.

4. The glucoamylase variant of claim 1, wherein the number of
substitutions is 1-20, e.g., 1-10 and 1-5, such as 1, 2, 3, 4, 5, 6, 7,
8, 9 or 10 substitutions.

7. The glucoamylase variant of claim 1, which has an improved property
relative to the parent, wherein the improved property is reduced
sensitivity to protease degradation.

8. A variant glucoamylase catalytic domain comprising a substitution at
least at a position corresponding to positions 79 of the mature
polypeptide of SEQ ID NO: 2, wherein the variant has glucoamylase
activity.

9. The variant glucoamylase catalytic domain of claim 8 selected from the
group consisting of: (a) a catalytic domain having at least 65% sequence
identity to amino acids 30 to 494 of SEQ ID NO: 2; (b) a catalytic domain
encoded by a polynucleotide that hybridizes under medium stringency
conditions with (i) nucleotides 88 to 1482 of SEQ ID NO: 1 or (ii) the
full-length complement of (i); (c) a catalytic domain encoded by a
polynucleotide having at least 65% sequence identity to (i) nucleotides
88 to 1482 of SEQ ID NO: 1; and (d) a variant of amino acids 30 to 494 of
SEQ ID NO: 2 comprising a substitution, deletion, and/or insertion at one
or more (e.g., several) positions; and wherein the catalytic domain has
glucoamylase activity.

10. A composition comprising the glucoamylase variant of claims 1.

11.-12. (canceled)

13. A process of producing a fermentation product from starch-containing
material comprising the steps of: (a) liquefying starch-containing
material in the presence of an alpha amylase; (b) saccharifying the
liquefied material; and (c) fermenting with a fermenting organism;
wherein step (a) and/or step (b) is carried out using at least a
glucoamylase variant of any of claim 1.

14. A process of producing a fermentation product from starch-containing
material, comprising the steps of: (a) saccharifying starch-containing
material at a temperature below the initial gelatinization temperature of
said starch-containing material; and (b) fermenting with a fermenting
organism, wherein step (a) is carried out using at least a glucoamylase
variant of claim 1.

16. A nucleic acid construct or expression vector comprising the
polynucleotide of claim 15 operably linked to one or more control
sequences that direct the production of the glucoamylase variant in an
expression host.

17. A recombinant host cell comprising the polynucleotide of claim 15
operably linked to one or more control sequences that direct the
production of the glucoamylase variant.

18. A method of producing a glucoamylase variant of claim 1, comprising:
(a) cultivating the host cell of claim 22 under conditions conducive for
production of the polypeptide; and (b) recovering the glucoamylase
variant.

Description:

[0001] This application contains a Sequence Listing in computer readable
form, which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to a glucoamylase variant,
polynucleotides encoding the variant, methods of producing the variants,
and methods of using the variants.

[0004] 2. Description of the Related Art

[0005] Glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) is an
enzyme, which catalyzes the release of D-glucose from the non-reducing
ends of starch or related oligo- and polysaccharide molecules.
Glucoamylases are produced by several filamentous fungi and yeast, with
those from Aspergillus being commercially most important.

[0006] Commercially, glucoamylases are used to convert starch, which is
already partially hydrolyzed by an alpha-amylase, to glucose. The glucose
may then be converted directly or indirectly into a fermentation product
using a fermenting organism. Examples of commercial fermentation products
include alcohols (e.g., ethanol, methanol, butanol, 1,3-propanediol);
organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic
acid, gluconic acid, gluconate, lactic acid, succinic acid,
2,5-diketo-D-gluconic acid); ketones (e.g., acetone); amino acids (e.g.,
glutamic acid); gases (e.g., H2 and CO2), and more complex
compounds, including, for example, antibiotics (e.g., penicillin and
tetracycline); enzymes; vitamins (e.g., riboflavin, B12,
beta-carotene); hormones, and other compounds which are difficult to
produce synthetically. Fermentation processes are also commonly used in
the consumable alcohol (e.g., beer and wine), dairy (e.g., in the
production of yogurt and cheese) industries.

[0007] The end product may also be syrup. For instance, the end product
may be glucose, but may also be converted, e.g., by glucose isomerase to
fructose or a mixture composed almost equally of glucose and fructose.
This mixture, or a mixture further enriched with fructose, is the most
commonly used high fructose corn syrup (HFCS) commercialized throughout
the world.

[0008] It is an object of the present invention to provide polypeptides
having glucoamylase activity and polynucleotides encoding the
polypeptides and which provide a high yield in fermentation product
production processes, such as ethanol production processes, including
one-step ethanol fermentation processes from un-gelatinized raw (or
uncooked) starch. Copending patent application, WO 2011/127802, discloses
a wild type glucoamylase from Penicillium oxalicum.

[0009] The present invention provides a glucoamylase variant with improved
properties compared to its parent.

SUMMARY OF THE INVENTION

[0010] The present invention relates to a glucoamylase variant, comprising
a substitution at least at a position corresponding to positions 79 of
the mature polypeptide of SEQ ID NO: 2, wherein the variant has
glucoamylase activity.

[0011] In further aspects the present invention relates to a variant
glucoamylase catalytic domain comprising a substitution at last at a
position corresponding to position 79 of the mature polypeptide of SEQ ID
NO: 2, wherein the variant has glucoamylase activity.

[0012] In another aspect the present invention relates to a composition
comprising the polypeptides of the invention.

[0013] The present invention also relates to isolated polynucleotides
encoding the variants; nucleic acid constructs, vectors, and host cells
comprising the polynucleotides; and methods of producing the variants.

[0014] The present invention also relates to methods of using the
polypeptides of the invention in production of syrup and/or a
fermentation product.

DEFINITIONS

[0015] Glucoamylase: The term glucoamylase (1,4-alpha-D-glucan
glucohydrolase, EC 3.2.1.3) is defined as an enzyme, which catalyzes the
release of D-glucose from the non-reducing ends of starch or related
oligo- and polysaccharide molecules. For purposes of the present
invention, glucoamylase activity is determined according to the procedure
described in the `Materials & Methods`-section herein.

[0016] The polypeptides of the present invention have at least 20%,
preferably at least 40%, preferably at least 45%, more preferably at
least 50%, more preferably at least 55%, more preferably at least 60%,
more preferably at least 65%, more preferably at least 70%, more
preferably at least 75%, more preferably at least 80%, more preferably at
least 85%, even more preferably at least 90%, most preferably at least
95%, and most preferably at least 100% of the glucoamylase activity of
the mature polypeptide of SEQ ID NO: 2.

[0017] Allelic variant: The term "allelic variant" means any of two or
more alternative forms of a gene occupying the same chromosomal locus.
Allelic variation arises naturally through mutation, and may result in
polymorphism within populations. Gene mutations can be silent (no change
in the encoded polypeptide) or may encode polypeptides having altered
amino acid sequences. An allelic variant of a polypeptide is a
polypeptide encoded by an allelic variant of a gene.

[0018] cDNA: The term "cDNA" means a DNA molecule that can be prepared by
reverse transcription from a mature, spliced, mRNA molecule obtained from
a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be
present in the corresponding genomic DNA. The initial, primary RNA
transcript is a precursor to mRNA that is processed through a series of
steps, including splicing, before appearing as mature spliced mRNA.

[0019] Coding sequence: The term "coding sequence" means a polynucleotide,
which directly specifies the amino acid sequence of a variant. The
boundaries of the coding sequence are generally determined by an open
reading frame, which begins with a start codon such as ATG, GTG or TTG
and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence
may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0020] Control sequences: The term "control sequences" means nucleic acid
sequences necessary for expression of a polynucleotide encoding a variant
of the present invention. Each control sequence may be native (i.e., from
the same gene) or foreign (i.e., from a different gene) to the
polynucleotide encoding the variant or native or foreign to each other.
Such control sequences include, but are not limited to, a leader,
polyadenylation sequence, propeptide sequence, promoter, signal peptide
sequence, and transcription terminator. At a minimum, the control
sequences include a promoter, and transcriptional and translational stop
signals. The control sequences may be provided with linkers for the
purpose of introducing specific restriction sites facilitating ligation
of the control sequences with the coding region of the polynucleotide
encoding a variant.

[0021] Expression: The term "expression" includes any step involved in the
production of a variant including, but not limited to, transcription,
post-transcriptional modification, translation, post-translational
modification, and secretion. Expression vector: The term "expression
vector" means a linear or circular DNA molecule that comprises a
polynucleotide encoding a variant and is operably linked to control
sequences that provide for its expression.

[0022] Fragment: The term "fragment" means a polypeptide having one or
more (e.g., several) amino acids absent from the amino and/or carboxyl
terminus of a mature polypeptide; wherein the fragment has glucoamylase
activity. In one aspect, a fragment contains at least 465 amino acid
residues (e.g., amino acids 30 to 494 of SEQ ID NO: 2).

[0023] High stringency conditions: The term "high stringency conditions"
means for probes of at least 100 nucleotides in length, prehybridization
and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200
micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide,
following standard Southern blotting procedures for 12 to 24 hours. The
carrier material is finally washed three times each for 15 minutes using
2×SSC, 0.2% SDS at 65° C.

[0024] Host cell: The term "host cell" means any cell type that is
susceptible to transformation, transfection, transduction, or the like
with a nucleic acid construct or expression vector comprising a
polynucleotide of the present invention. The term "host cell" encompasses
any progeny of a parent cell that is not identical to the parent cell due
to mutations that occur during replication.

[0025] Improved property: The term "improved property" means a
characteristic associated with a variant that is improved compared to the
parent. Such improved properties include, but are not limited to,
improved stability towards degradation or nicking by host proteases.
Improved stability is equivalent to reduced sensitivity. Preferably the
sensitivity is reduced by at least 10%, preferably at least 20%, more
preferably at least 30%, preferably at least 40%, preferably at least
45%, more preferably at least 50%, more preferably at least 55%, more
preferably at least 60%, more preferably at least 65%, more preferably at
least 70%, more preferably at least 75%, more preferably at least 80%,
more preferably at least 85%, even more preferably at least 90%, most
preferably at least 95%, and even most preferably at least 100%.

[0026] Isolated: The term "isolated" means a substance in a form or
environment which does not occur in nature. Non-limiting examples of
isolated substances include (1) any non-naturally occurring substance,
(2) any substance including, but not limited to, any enzyme, variant,
nucleic acid, protein, peptide or cofactor, that is at least partially
removed from one or more or all of the naturally occurring constituents
with which it is associated in nature; (3) any substance modified by the
hand of man relative to that substance found in nature; or (4) any
substance modified by increasing the amount of the substance relative to
other components with which it is naturally associated (e.g., multiple
copies of a gene encoding the substance; use of a stronger promoter than
the promoter naturally associated with the gene encoding the substance).
An isolated substance may be present in a fermentation broth sample.

[0027] Low stringency conditions: The term "low stringency conditions"
means for probes of at least 100 nucleotides in length, prehybridization
and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200
micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide,
following standard Southern blotting procedures for 12 to 24 hours. The
carrier material is finally washed three times each for 15 minutes using
2×SSC, 0.2% SDS at 50° C.

[0028] Mature polypeptide: The term "mature polypeptide" means a
polypeptide in its final form following translation and any
post-translational modifications, such as N-terminal processing,
C-terminal truncation, glycosylation, phosphorylation, etc. In one
aspect, the mature polypeptide is amino acids 22 to 616 of SEQ ID NO: 2
based on the program SignalP (Nielsen et al., 1997, Protein Engineering
10: 1-6) that predicts amino acids 1 to 21 of SEQ ID NO: 2 are a signal
peptide. It is known in the art that a host cell may produce a mixture of
two of more different mature polypeptides (i.e., with a different
C-terminal and/or N-terminal amino acid) expressed by the same
polynucleotide.

[0030] Medium stringency conditions: The term "medium stringency
conditions" means for probes of at least 100 nucleotides in length,
prehybridization and hybridization at 42° C. in 5×SSPE, 0.3%
SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35%
formamide, following standard Southern blotting procedures for 12 to 24
hours. The carrier material is finally washed three times each for 15
minutes using 2×SSC, 0.2% SDS at 55° C.

[0031] Medium-high stringency conditions: The term "medium-high stringency
conditions" means for probes of at least 100 nucleotides in length,
prehybridization and hybridization at 42° C. in 5×SSPE, 0.3%
SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and either
35% formamide, following standard Southern blotting procedures for 12 to
24 hours. The carrier material is finally washed three times each for 15
minutes using 2×SSC, 0.2% SDS at 60° C.

[0032] Mutant: The term "mutant" means a polynucleotide encoding a
variant. Nucleic acid construct: The term "nucleic acid construct" means
a nucleic acid molecule, either single- or double-stranded, which is
isolated from a naturally occurring gene or is modified to contain
segments of nucleic acids in a manner that would not otherwise exist in
nature or which is synthetic, which comprises one or more control
sequences.

[0033] Operably linked: The term "operably linked" means a configuration
in which a control sequence is placed at an appropriate position relative
to the coding sequence of a polynucleotide such that the control sequence
directs expression of the coding sequence.

[0034] Parent or parent glucoamylase: The term "parent" or "parent
glucoamylase" means a glucoamylase to which an alteration is made to
produce the enzyme variants of the present invention. The parent may be a
naturally occurring (wild-type) polypeptide or a variant or fragment
thereof. In one embodiment the parent glucoamylase is the mature
polypeptide of SEQ ID NO: 2.

[0035] Sequence identity: The relatedness between two amino acid sequences
or between two nucleotide sequences is described by the parameter
"sequence identity".

[0036] For purposes of the present invention, the sequence identity
between two amino acid sequences is determined using the Needleman-Wunsch
algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as
implemented in the Needle program of the EMBOSS package (EMBOSS: The
European Molecular Biology Open Software Suite, Rice et al., 2000, Trends
Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters
used are gap open penalty of 10, gap extension penalty of 0.5, and the
EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of
Needle labeled "longest identity" (obtained using the -nobrief option) is
used as the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment-Total Number of Gaps
in Alignment)

[0037] For purposes of the present invention, the sequence identity
between two deoxyribonucleotide sequences is determined using the
Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as
implemented in the Needle program of the EMBOSS package (EMBOSS: The
European Molecular Biology Open Software Suite, Rice et al., 2000,
supra), preferably version 5.0.0 or later. The parameters used are gap
open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL
(EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle
labeled "longest identity" (obtained using the -nobrief option) is used
as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment-Total
Number of Gaps in Alignment)

[0038] Subsequence: The term "subsequence" means a polynucleotide having
one or more (e.g., several) nucleotides absent from the 5' and/or 3' end
of a mature polypeptide coding sequence; wherein the subsequence encodes
a fragment having glucoamylase activity. In one aspect, a subsequence
contains at least 1395 nucleotides (e.g., nucleotides 88 to 1482 of SEQ
ID NO: 1)

[0039] Variant: The term "variant" means a polypeptide having glucoamylase
activity comprising an alteration, i.e., a substitution, insertion,
and/or deletion, at one or more (e.g., several) positions. A substitution
means replacement of the amino acid occupying a position with a different
amino acid; a deletion means removal of the amino acid occupying a
position; and an insertion means adding an amino acid adjacent to and
immediately following the amino acid occupying a position. The variants
of the present invention have at least 20%, e.g., at least 40%, at least
50%, at least 60%, at least 70%, at least 80%, at least 90%, at least
95%, or at least 100% of the glucoamylase activity of the mature
polypeptide of SEQ ID NO: 2.

[0040] Very high stringency conditions: The term "very high stringency
conditions" means for probes of at least 100 nucleotides in length,
prehybridization and hybridization at 42° C. in 5×SSPE, 0.3%
SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50%
formamide, following standard Southern blotting procedures for 12 to 24
hours. The carrier material is finally washed three times each for 15
minutes using 2×SSC, 0.2% SDS at 70° C.

[0041] Very low stringency conditions: The term "very low stringency
conditions" means for probes of at least 100 nucleotides in length,
prehybridization and hybridization at 42° C. in 5×SSPE, 0.3%
SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 25%
formamide, following standard Southern blotting procedures for 12 to 24
hours. The carrier material is finally washed three times each for 15
minutes using 2×SSC, 0.2% SDS at 45° C.

[0042] Wild-type glucoamylase: The term "wild-type" glucoamylase means a
glucoamylase expressed by a naturally occurring microorganism, such as a
bacterium, yeast, or filamentous fungus found in nature.

Conventions for Designation of Variants

[0043] For purposes of the present invention, the mature polypeptide
comprised in SEQ ID NO: 2 is used to determine the corresponding amino
acid residue in another glucoamylase. The amino acid sequence of another
glucoamylase is aligned with the mature polypeptide disclosed as amino
acids 22 to 616 of SEQ ID NO: 2, and based on the alignment, the amino
acid position number corresponding to any amino acid residue in the
mature polypeptide disclosed in SEQ ID NO: 2 is determined using the
Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:
443-453) as implemented in the Needle program of the EMBOSS package
(EMBOSS: The European Molecular Biology Open Software Suite, Rice et al.,
2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The
parameters used are gap open penalty of 10, gap extension penalty of 0.5,
and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
Accordingly, if e.g., the variant has a substitution at position 79, this
corresponds to position 100 in the full length polypeptide disclosed as
SEQ ID NO: 2, since amino acids 1-21 is the signal peptide, and position
22 will correspond to position 1 in the mature polypeptide.

[0045] When the other enzyme has diverged from the mature polypeptide of
SEQ ID NO: 2 such that traditional sequence-based comparison fails to
detect their relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295:
613-615), other pairwise sequence comparison algorithms can be used.
Greater sensitivity in sequence-based searching can be attained using
search programs that utilize probabilistic representations of polypeptide
families (profiles) to search databases. For example, the PSI-BLAST
program generates profiles through an iterative database search process
and is capable of detecting remote homologs (Atschul et al., 1997,
Nucleic Acids Res. 25: 3389-3402). Even greater sensitivity can be
achieved if the family or superfamily for the polypeptide has one or more
representatives in the protein structure databases. Programs such as
GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffin and Jones,
2003, Bioinformatics 19: 874-881) utilize information from a variety of
sources (PSI-BLAST, secondary structure prediction, structural alignment
profiles, and solvation potentials) as input to a neural network that
predicts the structural fold for a query sequence. Similarly, the method
of Gough et al., 2000, J. Mol. Biol. 313: 903-919, can be used to align a
sequence of unknown structure with the superfamily models present in the
SCOP database. These alignments can in turn be used to generate homology
models for the polypeptide, and such models can be assessed for accuracy
using a variety of tools developed for that purpose.

[0046] For proteins of known structure, several tools and resources are
available for retrieving and generating structural alignments. For
example the SCOP superfamilies of proteins have been structurally
aligned, and those alignments are accessible and downloadable. Two or
more protein structures can be aligned using a variety of algorithms such
as the distance alignment matrix (Holm and Sander, 1998, Proteins 33:
88-96) or combinatorial extension (Shindyalov and Bourne, 1998, Protein
Engineering 11: 739-747), and implementation of these algorithms can
additionally be utilized to query structure databases with a structure of
interest in order to discover possible structural homologs (e.g., Holm
and Park, 2000, Bioinformatics 16: 566-567).

[0047] In describing the variants of the present invention, the
nomenclature described below is adapted for ease of reference. The
accepted IUPAC single letter or three letter amino acid abbreviation is
employed.

[0048] Substitutions. For an amino acid substitution, the following
nomenclature is used: Original amino acid, position, substituted amino
acid. Accordingly, the substitution of threonine at position 226 with
alanine is designated as "Thr226Ala" or "T226A". Multiple mutations are
separated by addition marks ("+"), e.g., "Gly205Arg+Ser411Phe" or
"G205R+5411 F", representing substitutions at positions 205 and 411 of
glycine (G) with arginine (R) and serine (S) with phenylalanine (F),
respectively.

[0049] Deletions.

[0050] For an amino acid deletion, the following nomenclature is used:
Original amino acid, position, *. Accordingly, the deletion of glycine at
position 195 is designated as "Gly195*" or "G195*". Multiple deletions
are separated by addition marks ("+"), e.g., "Gly195*+Ser411*" or
"G195*+S411*".

[0051] Insertions.

[0052] For an amino acid insertion, the following nomenclature is used:
Original amino acid, position, original amino acid, inserted amino acid.
Accordingly the insertion of lysine after glycine at position 195 is
designated "Glyl95GlyLys" or "G195GK". An insertion of multiple amino
acids is designated "Original amino acid, position, original amino acid,
inserted amino acid #1, inserted amino acid #2"; etc. For example, the
insertion of lysine and alanine after glycine at position 195 is
indicated as "Glyl95GlyLysAla" or "G195GKA".

[0053] In such cases the inserted amino acid residue(s) are numbered by
the addition of lower case letters to the position number of the amino
acid residue preceding the inserted amino acid residue(s). In the above
example, the sequence would thus be:

TABLE-US-00001
Parent: Variant:
195 195 195a 195b
G G - K - A

[0054] Multiple Alterations.

[0055] Variants comprising multiple alterations are separated by addition
marks ("+"), e.g., "Arg170Tyr+Glyl95Glu" or "R170Y+G195E" representing a
substitution of arginine and glycine at positions 170 and 195 with
tyrosine and glutamic acid, respectively.

[0056] Different Alterations.

[0057] Where different alterations can be introduced at a position, the
different alterations are separated by a comma, e.g., "Arg170Tyr, Glu"
represents a substitution of arginine at position 170 with tyrosine or
glutamic acid. Thus, "Tyr167Gly, Ala+Arg170Gly, Ala" designates the
following variants: "Tyr167Gly+Arg170Gly", "Tyr167Gly+Arg170Ala",
"Tyr167Ala+Arg170Gly", and "Tyr167Ala+Arg170Ala".

DETAILED DESCRIPTION OF THE INVENTION

[0058] The present invention relates to isolated glucoamylase variants,
comprising a substitution at least at a position corresponding to
position 79, of the mature polypeptide of SEQ ID NO: 2, wherein the
variant has glucoamylase activity.

Variants

[0059] The present invention also provides glucoamylase variants,
comprising a substitution at least at a position corresponding to
positions 79 of the mature polypeptide of SEQ ID NO: 2, wherein the
variant has glucoamylase activity. The variants according to the
invention has reduced sensitivity to protease degradation.

[0060] In a further embodiment the variant is selected from the group
consisting of:

[0061] a) a polypeptide having at least 65% sequence identity to the
mature polypeptide of SEQ ID NO: 2;

[0062] b) a polypeptide encoded by a polynucleotide that hybridizes under
low stringency conditions with (i) the mature polypeptide coding sequence
of SEQ ID NO: 1, or (ii) the full-length complement of (i);

[0063] c) a polypeptide encoded by a polynucleotide having at least 65%
identity to the mature polypeptide coding sequence of SEQ ID NO: 1; and

[0064] d) a fragment of the mature polypeptide of SEQ ID NO: 2, which has
glucoamylase activity.

[0065] In an embodiment, the variant has a sequence identity of at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less
than 100%, to the amino acid sequence of the mature parent glucoamylase.

[0066] In another embodiment, the variant has at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, such as at least
96%, at least 97%, at least 98%, or at least 99%, but less than 100%,
sequence identity to the mature polypeptide of SEQ ID NO: 2.

[0067] In one aspect, the number of alterations in the variants of the
present invention is 1-20, e.g., 1-10 and 1-5, such as 1, 2, 3, 4, 5, 6,
7, 8, 9 or 10 alterations.

[0069] In another aspect, the variant is encoded by a polynucleotide
having at least 65%, at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99% sequence identity to the mature polypeptide coding
sequence of SEQ ID NO: 1.

[0070] In another aspect, the variant comprises or consists of a
substitution at a position corresponding to position 79. In another
aspect, the amino acid at a position corresponding to position 79 is
substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu,
Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In another aspect, the amino
acid at a position corresponding to position 79 is substituted with Ala,
Gly, Ile, Leu, Ser, Thr, Val, preferably Val. In another aspect, the
variant comprises or consists of the substitution selected from K79V,
K79A, K79G, K79I, K79L, K79S, K79T of the mature polypeptide of SEQ ID
NO: 2. In another aspect, the variant comprises or consists of the
substitution K79V of the mature polypeptide of SEQ ID NO: 2. In a further
specific embodiment the mature variant polypeptide consists of SEQ ID NO:
3.

[0071] The variants may further comprise one or more additional
substitutions at one or more (e.g., several) other positions.

[0072] The amino acid changes may be of a minor nature, that is
conservative amino acid substitutions or insertions that do not
significantly affect the folding and/or activity of the protein; small
deletions, typically of 1-30 amino acids; small amino- or
carboxyl-terminal extensions, such as an amino-terminal methionine
residue; a small linker peptide of up to 20-25 residues; or a small
extension that facilitates purification by changing net charge or another
function, such as a poly-histidine tract, an antigenic epitope or a
binding domain.

[0074] Alternatively, the amino acid changes are of such a nature that the
physico-chemical properties of the polypeptides are altered. For example,
amino acid changes may improve the thermal stability of the polypeptide,
alter the substrate specificity, change the pH optimum, and the like.

[0075] Essential amino acids in a polypeptide can be identified according
to procedures known in the art, such as site-directed mutagenesis or
alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:
1081-1085). In the latter technique, single alanine mutations are
introduced at every residue in the molecule, and the resultant mutant
molecules are tested for glucoamylase activity to identify amino acid
residues that are critical to the activity of the molecule. See also,
Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site of
the enzyme or other biological interaction can also be determined by
physical analysis of structure, as determined by such techniques as
nuclear magnetic resonance, crystallography, electron diffraction, or
photoaffinity labeling, in conjunction with mutation of putative contact
site amino acids. See, for example, de Vos et al., 1992, Science 255:
306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al.,
1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can
also be inferred from an alignment with a related polypeptide.

[0076] In one embodiment the variants may consist of at least the
catalytic domain of 465 amino acids, e.g. amino acids 30 to 494 in the
parent glucoamylase shown as SEQ ID NO: 2.

[0077] A second aspect of the present invention relates to a variant
glucoamylase catalytic domain comprising a substitution at least at a
position corresponding to positions 79 of the mature polypeptide of SEQ
ID NO: 2, wherein the variant has glucoamylase activity.

[0078] The variant glucoamylase catalytic domain may in one embodiment be
selected from the group consisting of:

[0080] (b) a catalytic domain encoded by a polynucleotide that hybridizes
under medium stringency conditions with (i) nucleotides 88 to 1482 of SEQ
ID NO: 1 or (ii) the full-length complement of (i);

[0081] (c) a catalytic domain encoded by a polynucleotide having at least
65% sequence identity to (i) nucleotides 88 to 1482 of SEQ ID NO: 1; and

[0082] (d) a variant of amino acids 30 to 494 of SEQ ID NO: 2 comprising a
substitution, deletion, and/or insertion at one or more (e.g., several)
positions; and wherein the catalytic domain has glucoamylase activity.

[0083] In one embodiment the catalytic domain may be considered to include
the linker region from amino acids 495 to 506 of SEQ ID NO: 2. Amino
acids 507 to 615 of SEQ ID NO: 2 correspond to a starch binding domain.
In one embodiment the catalytic domain according to the invention is
connected to a linker and a carbohydrate binding domain.

[0084] In an embodiment, the variant glucoamylase catalytic domain has
sequence identity of at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, or at least 99%, but less than 100%, to the amino acid sequence of
the parent glucoamylase catalytic domain.

[0085] In another embodiment, the variant has at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, such as at least
96%, at least 97%, at least 98%, or at least 99%, but less than 100%,
sequence identity to the catalytic domain comprised in SEQ ID NO: 2, e.g.
amino acids 30 to 494 of SEQ ID NO: 2.

[0087] In an embodiment, the variant has improved chemical stability
compared to the parent enzyme. In particular the improved stability is
improved stability towards nicking by host proteases. Thus in one
embodiment the the invention relates to a variant which has an improved
property relative to the parent, wherein the improved property is reduced
sensitivity to protease degradation.

Parent Glucoamylases

[0088] The parent glucoamylase may be (a) a polypeptide having at least
65% sequence identity to the mature polypeptide of SEQ ID NO: 2; (b) a
polypeptide encoded by a polynucleotide that hybridizes under low
stringency conditions with (i) the mature polypeptide coding sequence of
SEQ ID NO: 1, or (ii) the full-length complement of (i); or (c) a
polypeptide encoded by a polynucleotide having at least 65% sequence
identity to the mature polypeptide coding sequence of SEQ ID NO: 1; or d)
a fragment of the mature polypeptide of SEQ ID NO: 2, which has
glucoamylase activity.

[0089] In an aspect, the parent has a sequence identity to the mature
polypeptide of SEQ ID NO: 2 of at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 98%, at least 99%, or 100%, which have glucoamylase activity. In
one aspect, the amino acid sequence of the parent differs by no more than
10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9, from the mature
polypeptide of SEQ ID NO: 2.

[0090] In another aspect, the parent comprises or consists of the amino
acid sequence of SEQ ID NO: 2. In another aspect, the parent comprises or
consists of the mature polypeptide of SEQ ID NO: 2. In another aspect,
the parent comprises or consists of amino acids 30 to 494 of SEQ ID NO:
2.

[0091] In another aspect, the parent is a fragment of the mature
polypeptide of SEQ ID NO: 2 containing at least 465 amino acid residues,
e.g., at least 470 and at least 475 amino acid residues.

[0092] In another embodiment, the parent is an allelic variant of the
mature polypeptide of SEQ ID NO: 2.

[0094] The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as
well as the polypeptide of SEQ ID NO: 2 or a fragment thereof, may be
used to design nucleic acid probes to identify and clone DNA encoding a
parent from strains of different genera or species according to methods
well known in the art. In particular, such probes can be used for
hybridization with the genomic DNA or cDNA of a cell of interest,
following standard Southern blotting procedures, in order to identify and
isolate the corresponding gene therein. Such probes can be considerably
shorter than the entire sequence, but should be at least 15, e.g., at
least 25, at least 35, or at least 70 nucleotides in length. Preferably,
the nucleic acid probe is at least 100 nucleotides in length, e.g., at
least 200 nucleotides, at least 300 nucleotides, at least 400
nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least
700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in
length. Both DNA and RNA probes can be used. The probes are typically
labeled for detecting the corresponding gene (for example, with 32P,
3H, 35S, biotin, or avidin). Such probes are encompassed by the
present invention.

[0095] A genomic DNA or cDNA library prepared from such other strains may
be screened for DNA that hybridizes with the probes described above and
encodes a parent. Genomic or other DNA from such other strains may be
separated by agarose or polyacrylamide gel electrophoresis, or other
separation techniques. DNA from the libraries or the separated DNA may be
transferred to and immobilized on nitrocellulose or other suitable
carrier material. In order to identify a clone or DNA that hybridizes
with SEQ ID NO: 1 or a subsequence thereof, the carrier material is used
in a Southern blot.

[0096] For purposes of the present invention, hybridization indicates that
the polynucleotide hybridizes to a labeled nucleic acid probe
corresponding to (i) SEQ ID NO: 1; (ii) the mature polypeptide coding
sequence of SEQ ID NO: 1; (iii) the full-length complement thereof; or
(iv) a subsequence thereof; under very low to very high stringency
conditions. Molecules to which the nucleic acid probe hybridizes under
these conditions can be detected using, for example, X-ray film or any
other detection means known in the art.

[0097] In one aspect, the nucleic acid probe is the mature polypeptide
coding sequence of SEQ ID NO: 1. In another aspect, the nucleic acid
probe is nucleotides 64 to 1848 of SEQ ID NO: 1. In another aspect, the
nucleic acid probe is a polynucleotide that encodes the polypeptide of
SEQ ID NO: 2; the mature polypeptide thereof; or a fragment thereof. In
another aspect, the nucleic acid probe is SEQ ID NO: 1.

[0098] In another embodiment, the parent is encoded by a polynucleotide
having a sequence identity to the mature polypeptide coding sequence of
SEQ ID NO: 1 of at least 65%, at least 70%, at least 75%, at least 80%,
at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100%.

[0099] The polypeptide may be a hybrid polypeptide in which a region of
one polypeptide is fused at the N-terminus or the C-terminus of a region
of another polypeptide.

[0100] The parent may be a fusion polypeptide or cleavable fusion
polypeptide in which another polypeptide is fused at the N-terminus or
the C-terminus of the polypeptide of the present invention. A fusion
polypeptide is produced by fusing a polynucleotide encoding another
polypeptide to a polynucleotide of the present invention. Techniques for
producing fusion polypeptides are known in the art, and include ligating
the coding sequences encoding the polypeptides so that they are in frame
and that expression of the fusion polypeptide is under control of the
same promoter(s) and terminator. Fusion polypeptides may also be
constructed using intein technology in which fusion polypeptides are
created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583;
Dawson et al., 1994, Science 266: 776-779).

[0102] In one particular embodiment the hybrid polypeptide comprises the
variant glucoamylase catalytic domain fused to a linker and a
carbohydrate binding domain.

[0103] The parent may be obtained from microorganisms of any genus. For
purposes of the present invention, the term "obtained from" as used
herein in connection with a given source shall mean that the parent
encoded by a polynucleotide is produced by the source or by a strain in
which the polynucleotide from the source has been inserted. In one
aspect, the parent is secreted extracellularly.

[0104] The parent may be a fungal glucoamylase. For example, the parent
may be a Penicillium glucoamylase such as, e.g., a Penicillium oxalicum
glucoamylase.

[0105] In another aspect, the parent is a Penicillium oxalicum, e.g., the
glucoamylase of SEQ ID NO: 2 or the mature polypeptide thereof.

[0106] It will be understood that for the aforementioned species, the
invention encompasses both the perfect and imperfect states, and other
taxonomic equivalents, e.g., anamorphs, regardless of the species name by
which they are known. Those skilled in the art will readily recognize the
identity of appropriate equivalents.

[0107] Strains of these species are readily accessible to the public in a
number of culture collections, such as the American Type Culture
Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen
GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural
Research Service Patent Culture Collection, Northern Regional Research
Center (NRRL).

[0108] The parent may be identified and obtained from other sources
including microorganisms isolated from nature (e.g., soil, composts,
water, etc.) or DNA samples obtained directly from natural materials
(e.g., soil, composts, water, etc.) using the above-mentioned probes.
Techniques for isolating microorganisms and DNA directly from natural
habitats are well known in the art. A polynucleotide encoding a parent
may then be obtained by similarly screening a genomic DNA or cDNA library
of another microorganism or mixed DNA sample. Once a polynucleotide
encoding a parent has been detected with the probe(s), the polynucleotide
can be isolated or cloned by utilizing techniques that are known to those
of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

Preparation of Variants

[0109] The variants can be prepared using any mutagenesis procedure known
in the art, such as site-directed mutagenesis, synthetic gene
construction, semi-synthetic gene construction, random mutagenesis,
shuffling, etc.

[0110] Site-directed mutagenesis is a technique in which one or more
(e.g., several) mutations are introduced at one or more defined sites in
a polynucleotide encoding the parent.

[0111] Site-directed mutagenesis can be accomplished in vitro by PCR
involving the use of oligonucleotide primers containing the desired
mutation. Site-directed mutagenesis can also be performed in vitro by
cassette mutagenesis involving the cleavage by a restriction enzyme at a
site in the plasmid comprising a polynucleotide encoding the parent and
subsequent ligation of an oligonucleotide containing the mutation in the
polynucleotide. Usually the restriction enzyme that digests the plasmid
and the oligonucleotide is the same, permitting sticky ends of the
plasmid and the insert to ligate to one another. See, e.g., Scherer and
Davis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton et al.,
1990, Nucleic Acids Res. 18: 7349-4966.

[0113] Any site-directed mutagenesis procedure can be used in the present
invention. There are many commercial kits available that can be used to
prepare variants.

[0114] Synthetic gene construction entails in vitro synthesis of a
designed polynucleotide molecule to encode a polypeptide of interest.
Gene synthesis can be performed utilizing a number of techniques, such as
the multiplex microchip-based technology described by Tian et al. (2004,
Nature 432: 1050-1054) and similar technologies wherein oligonucleotides
are synthesized and assembled upon photo-programmable microfluidic chips.

[0116] Mutagenesis/shuffling methods can be combined with high-throughput,
automated screening methods to detect activity of cloned, mutagenized
polypeptides expressed by host cells (Ness et al., 1999, Nature
Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active
polypeptides can be recovered from the host cells and rapidly sequenced
using standard methods in the art. These methods allow the rapid
determination of the importance of individual amino acid residues in a
polypeptide.

[0117] Semi-synthetic gene construction is accomplished by combining
aspects of synthetic gene construction, and/or site-directed mutagenesis,
and/or random mutagenesis, and/or shuffling. Semi-synthetic construction
is typified by a process utilizing polynucleotide fragments that are
synthesized, in combination with PCR techniques. Defined regions of genes
may thus be synthesized de novo, while other regions may be amplified
using site-specific mutagenic primers, while yet other regions may be
subjected to error-prone PCR or non-error prone PCR amplification.
Polynucleotide subsequences may then be shuffled.

Polynucleotides

[0118] The present invention also relates to isolated polynucleotides
encoding a variant of the present invention.

Nucleic Acid Constructs

[0119] The present invention also relates to nucleic acid constructs
comprising a polynucleotide encoding a variant of the present invention
operably linked to one or more control sequences that direct the
expression of the coding sequence in a suitable host cell under
conditions compatible with the control sequences.

[0120] The polynucleotide may be manipulated in a variety of ways to
provide for expression of a variant. Manipulation of the polynucleotide
prior to its insertion into a vector may be desirable or necessary
depending on the expression vector. The techniques for modifying
polynucleotides utilizing recombinant DNA methods are well known in the
art.

[0121] The control sequence may be a promoter, a polynucleotide which is
recognized by a host cell for expression of the polynucleotide. The
promoter contains transcriptional control sequences that mediate the
expression of the variant. The promoter may be any polynucleotide that
shows transcriptional activity in the host cell including mutant,
truncated, and hybrid promoters, and may be obtained from genes encoding
extracellular or intracellular polypeptides either homologous or
heterologous to the host cell.

[0125] The control sequence may also be a transcription terminator, which
is recognized by a host cell to terminate transcription. The terminator
sequence is operably linked to the 3'-terminus of the polynucleotide
encoding the variant. Any terminator that is functional in the host cell
may be used.

[0131] The control sequence may also be a leader, a nontranslated region
of an mRNA that is important for translation by the host cell. The leader
sequence is operably linked to the 5'-terminus of the polynucleotide
encoding the variant. Any leader that is functional in the host cell may
be used.

[0134] The control sequence may also be a polyadenylation sequence, a
sequence operably linked to the 3'-terminus of the variant-encoding
sequence and, when transcribed, is recognized by the host cell as a
signal to add polyadenosine residues to transcribed mRNA. Any
polyadenylation sequence that is functional in the host cell may be used.

[0137] The control sequence may also be a signal peptide coding region
that encodes a signal peptide linked to the N-terminus of a variant and
directs the variant into the cell's secretory pathway. The 5'-end of the
coding sequence of the polynucleotide may inherently contain a signal
peptide coding sequence naturally linked in translation reading frame
with the segment of the coding sequence that encodes the variant.
Alternatively, the 5'-end of the coding sequence may contain a signal
peptide coding sequence that is foreign to the coding sequence. A foreign
signal peptide coding sequence may be required where the coding sequence
does not naturally contain a signal peptide coding sequence.
Alternatively, a foreign signal peptide coding sequence may simply
replace the natural signal peptide coding sequence in order to enhance
secretion of the variant. However, any signal peptide coding sequence
that directs the expressed variant into the secretory pathway of a host
cell may be used.

[0141] The control sequence may also be a propeptide coding sequence that
encodes a propeptide positioned at the N-terminus of a variant. The
resultant polypeptide is known as a proenzyme or propolypeptide (or a
zymogen in some cases). A propolypeptide is generally inactive and can be
converted to an active polypeptide by catalytic or autocatalytic cleavage
of the propeptide from the propolypeptide. The propeptide coding sequence
may be obtained from the genes for Bacillus subtilis alkaline protease
(aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora
thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase,
and Saccharomyces cerevisiae alpha-factor.

[0142] Where both signal peptide and propeptide sequences are present, the
propeptide sequence is positioned next to the N-terminus of the variant
and the signal peptide sequence is positioned next to the N-terminus of
the propeptide sequence.

[0143] It may also be desirable to add regulatory sequences that regulate
expression of the variant relative to the growth of the host cell.
Examples of regulatory systems are those that cause expression of the
gene to be turned on or off in response to a chemical or physical
stimulus, including the presence of a regulatory compound. Regulatory
systems in prokaryotic systems include the lac, tac, and trp operator
systems. In yeast, the ADH2 system or GAL1 system may be used. In
filamentous fungi, the Aspergillus niger glucoamylase promoter,
Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzae
glucoamylase promoter may be used. Other examples of regulatory sequences
are those that allow for gene amplification. In eukaryotic systems, these
regulatory sequences include the dihydrofolate reductase gene that is
amplified in the presence of methotrexate, and the metallothionein genes
that are amplified with heavy metals. In these cases, the polynucleotide
encoding the variant would be operably linked with the regulatory
sequence.

Expression Vectors

[0144] The present invention also relates to recombinant expression
vectors comprising a polynucleotide encoding a variant of the present
invention, a promoter, and transcriptional and translational stop
signals. The various nucleotide and control sequences may be joined
together to produce a recombinant expression vector that may include one
or more convenient restriction sites to allow for insertion or
substitution of the polynucleotide encoding the variant at such sites.
Alternatively, the polynucleotide may be expressed by inserting the
polynucleotide or a nucleic acid construct comprising the polynucleotide
into an appropriate vector for expression. In creating the expression
vector, the coding sequence is located in the vector so that the coding
sequence is operably linked with the appropriate control sequences for
expression.

[0145] The recombinant expression vector may be any vector (e.g., a
plasmid or virus) that can be conveniently subjected to recombinant DNA
procedures and can bring about expression of the polynucleotide. The
choice of the vector will typically depend on the compatibility of the
vector with the host cell into which the vector is to be introduced. The
vector may be a linear or closed circular plasmid.

[0146] The vector may be an autonomously replicating vector, i.e., a
vector that exists as an extrachromosomal entity, the replication of
which is independent of chromosomal replication, e.g., a plasmid, an
extrachromosomal element, a minichromosome, or an artificial chromosome.
The vector may contain any means for assuring self-replication.
Alternatively, the vector may be one that, when introduced into the host
cell, is integrated into the genome and replicated together with the
chromosome(s) into which it has been integrated. Furthermore, a single
vector or plasmid or two or more vectors or plasmids that together
contain the total DNA to be introduced into the genome of the host cell,
or a transposon, may be used.

[0147] The vector preferably contains one or more selectable markers that
permit easy selection of transformed, transfected, transduced, or the
like cells. A selectable marker is a gene the product of which provides
for biocide or viral resistance, resistance to heavy metals, prototrophy
to auxotrophs, and the like.

[0149] The vector preferably contains an element(s) that permits
integration of the vector into the host cell's genome or autonomous
replication of the vector in the cell independent of the genome.

[0150] For integration into the host cell genome, the vector may rely on
the polynucleotide's sequence encoding the variant or any other element
of the vector for integration into the genome by homologous or
non-homologous recombination. Alternatively, the vector may contain
additional polynucleotides for directing integration by homologous
recombination into the genome of the host cell at a precise location(s)
in the chromosome(s). To increase the likelihood of integration at a
precise location, the integrational elements should contain a sufficient
number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000
base pairs, and 800 to 10,000 base pairs, which have a high degree of
sequence identity to the corresponding target sequence to enhance the
probability of homologous recombination. The integrational elements may
be any sequence that is homologous with the target sequence in the genome
of the host cell. Furthermore, the integrational elements may be
non-encoding or encoding polynucleotides. On the other hand, the vector
may be integrated into the genome of the host cell by non-homologous
recombination.

[0151] For autonomous replication, the vector may further comprise an
origin of replication enabling the vector to replicate autonomously in
the host cell in question. The origin of replication may be any plasmid
replicator mediating autonomous replication that functions in a cell. The
term "origin of replication" or "plasmid replicator" means a
polynucleotide that enables a plasmid or vector to replicate in vivo.

[0152] Examples of bacterial origins of replication are the origins of
replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting
replication in E. coli, and pUB110, pE194, pTA1060, and pAMR1 permitting
replication in Bacillus.

[0153] Examples of origins of replication for use in a yeast host cell are
the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1
and CEN3, and the combination of ARS4 and CEN6.

[0154] Examples of origins of replication useful in a filamentous fungal
cell are AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,
1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of the
AMA1 gene and construction of plasmids or vectors comprising the gene can
be accomplished according to the methods disclosed in WO 00/24883.

[0155] More than one copy of a polynucleotide of the present invention may
be inserted into a host cell to increase production of a variant. An
increase in the copy number of the polynucleotide can be obtained by
integrating at least one additional copy of the sequence into the host
cell genome or by including an amplifiable selectable marker gene with
the polynucleotide where cells containing amplified copies of the
selectable marker gene, and thereby additional copies of the
polynucleotide, can be selected for by cultivating the cells in the
presence of the appropriate selectable agent.

[0156] The procedures used to ligate the elements described above to
construct the recombinant expression vectors of the present invention are
well known to one skilled in the art (see, e.g., Sambrook et al., 1989,
supra).

Host Cells

[0157] The present invention also relates to recombinant host cells,
comprising a polynucleotide encoding a variant of the present invention
operably linked to one or more control sequences that direct the
production of a variant of the present invention. A construct or vector
comprising a polynucleotide is introduced into a host cell so that the
construct or vector is maintained as a chromosomal integrant or as a
self-replicating extra-chromosomal vector as described earlier. The term
"host cell" encompasses any progeny of a parent cell that is not
identical to the parent cell due to mutations that occur during
replication. The choice of a host cell will to a large extent depend upon
the gene encoding the variant and its source.

[0158] The host cell may be any cell useful in the recombinant production
of a variant, e.g., a prokaryote or a eukaryote.

[0164] The host cell may also be a eukaryote, such as a mammalian, insect,
plant, or fungal cell.

[0165] The host cell may be a fungal cell. "Fungi" as used herein includes
the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as
well as the Oomycota and all mitosporic fungi (as defined by Hawksworth
et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,
1995, CAB International, University Press, Cambridge, UK).

[0166] The fungal host cell may be a yeast cell. "Yeast" as used herein
includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast,
and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the
classification of yeast may change in the future, for the purposes of
this invention, yeast shall be defined as described in Biology and
Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App.
Bacteriol. Symposium Series No. 9, 1980).

[0168] The fungal host cell may be a filamentous fungal cell. "Filamentous
fungi" include all filamentous forms of the subdivision Eumycota and
Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous
fungi are generally characterized by a mycelial wall composed of chitin,
cellulose, glucan, chitosan, mannan, and other complex polysaccharides.
Vegetative growth is by hyphal elongation and carbon catabolism is
obligately aerobic. In contrast, vegetative growth by yeasts such as
Saccharomyces cerevisiae is by budding of a unicellular thallus and
carbon catabolism may be fermentative.

[0172] The present invention also relates to methods of producing a
variant, comprising: (a) cultivating a host cell of the present invention
under conditions suitable for expression of the variant; and (b)
recovering the variant.

[0173] The host cells are cultivated in a nutrient medium suitable for
production of the variant using methods known in the art. For example,
the cell may be cultivated by shake flask cultivation, or small-scale or
large-scale fermentation (including continuous, batch, fed-batch, or
solid state fermentations) in laboratory or industrial fermentors
performed in a suitable medium and under conditions allowing the variant
to be expressed and/or isolated. The cultivation takes place in a
suitable nutrient medium comprising carbon and nitrogen sources and
inorganic salts, using procedures known in the art. Suitable media are
available from commercial suppliers or may be prepared according to
published compositions (e.g., in catalogues of the American Type Culture
Collection). If the variant is secreted into the nutrient medium, the
variant can be recovered directly from the medium. If the variant is not
secreted, it can be recovered from cell lysates.

[0174] The variant may be detected using methods known in the art that are
specific for the variants. These detection methods include, but are not
limited to, use of specific antibodies, formation of an enzyme product,
or disappearance of an enzyme substrate. For example, an enzyme assay may
be used to determine the activity of the variant.

[0175] The variant may be recovered using methods known in the art. For
example, the variant may be recovered from the nutrient medium by
conventional procedures including, but not limited to, collection,
centrifugation, filtration, extraction, spray-drying, evaporation, or
precipitation.

[0177] In an alternative aspect, the variant is not recovered, but rather
a host cell of the present invention expressing the variant is used as a
source of the variant.

Compositions

[0178] The present invention also relates to compositions comprising a
polypeptide of the present invention. Preferably the composition also
comprises a carrier and/or an excipient. More preferably, the
compositions are enriched in such a polypeptide. The term "enriched"
indicates that the glucoamylase activity of the composition has been
increased, e.g., with an enrichment factor of at least 1.1. Preferably,
the compositions are formulated to provide desirable characteristics such
as low color, low odor and acceptable storage stability.

[0182] The polypeptide compositions may be prepared in accordance with
methods known in the art and may be in the form of a liquid or a dry
composition. For instance, the polypeptide composition may be in the form
of a granulate or a microgranulate. The polypeptide to be included in the
composition may be stabilized in accordance with methods known in the
art.

[0183] Examples are given below of preferred uses of the polypeptide or
polypeptide compositions of the invention. The dosage of the polypeptide
composition of the invention and other conditions under which the
composition is used may be determined on the basis of methods known in
the art.

Combination of glucoamylase and alpha-amylase

[0184] According to this aspect of the invention a glucoamylase of the
invention may be combined with an alpha-amylase. Preferably, the ratio of
acid alpha-amylase to glucoamylase is between 0.05 and 5.0 AFAU/AGU. More
preferably the ratio between acid alpha-amylase activity and glucoamylase
activity is at least 0.10, at least 0.15, at least 0.20, at least 0.25,
at least 0.30, at least 0.35, at least 0.40, at least 0.45, at least
0.50, at least 0.55, at least 0.60, at least 0.65, at least 0.70, at
least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.95,
at least 1.00, at least 1.05, at least 1.10, at least 1.20, at least
1.30, at least 1.40, at least 1.50, at least 1.60, at least 1.70, at
least 1.80, at least 1.85, or even at least 1.90 AFAU/AGU. However, the
ratio between acid alpha-amylase activity and glucoamylase activity
should preferably be less than 4.50, less than 4.00, less than 3.50, less
than 3.00, less than 2.50, or even less than 2.25 AFAU/AGU.

[0185] Above composition is suitable for use in liquefaction,
saccharification, and/or fermentation process, preferably in starch
conversion, especially for producing syrup and fermentation products,
such as ethanol.

[0186] Examples are given below of preferred uses of the polypeptide
compositions of the present invention. The dosage of the polypeptide
composition of the invention and other conditions under which the
composition is used may be determined on the basis of methods known in
the art.

Uses

[0187] The present invention is also directed to use of a polypeptide of
the present invention in a liquefaction, a saccharification and/or a
fermentation process. The polypeptide may be used in a single process,
for example, in a liquefaction process, a saccharification process, or a
fermentation process. The polypeptide may also be used in a combination
of processes for example in a liquefaction and saccharification process,
in a liquefaction and fermentation process, or in a saccharification and
fermentation process, preferably in relation to starch conversion.

[0188] In a preferred aspect of the present invention, the liquefaction,
saccharification and/or fermentation process includes sequentially or
simultaneously performed liquefaction and saccharification processes.

[0189] In conventional enzymatic liquefaction process, thermostable
alpha-amylase is added and the long chained starch is degraded into
branched and linear shorter units (maltodextrins), but glucoamylase is
not added.

[0190] When applying the glucoamylase of the present invention,
potentially in combination with an alpha-amylase in a liquefaction and/or
saccharification process, especially in a simultaneous liquefaction and
saccharification process, the process can be conducted at a higher
temperature. By conducting the liquefaction and/or saccharification
processs at higher temperatures the process can be carried out in a
shorter period of time or alternatively the process can be carried out
using lower enzyme dosage. Furthermore, the risk of microbial
contamination is reduced when carrying the liquefaction and/or
saccharification process at higher temperature.

Conversion of Starch-Containing Material

[0191] The present invention provides a use of the glucoamylase of the
invention for producing glucoses and the like from starch. Generally, the
method includes the steps of partially hydrolyzing precursor starch using
glucoamylase of the present invention either alone or in the presence of
an alpha-amylase.

[0192] The glucoamylase of the invention may also be used in combination
with an enzyme that hydrolyzes only alpha-(1,6)-glucosidic bonds in
molecules comprising at least four glucosyl residues.

[0193] In a further aspect the invention relates to the use of a
glucoamylase of the invention in starch conversion. Furthermore, the
glucoamylase of the invention may be used in a continuous starch
conversion process including a continuous saccharification process.

Production of Syrup, Beverage and/or Fermentation Product

[0194] Uses of the glucoamylase of the invention include conversion of
starch to e.g., syrup beverage, and/or a fermentation product, including
ethanol.

[0195] The present invention also provides a process of using a
glucoamylase of the invention for producing syrup, such as glucose and
the like, from starch-containing material. Suitable starting materials
are exemplified in the "Starch-containing materials"-section. Generally,
the process comprises the steps of partially or totally hydrolyzing
starch-containing material (liquefaction and/or saccharification) in the
presence of the glucoamylase of the present invention alone or in
combination with alpha-amylase to release glucose from the non-reducing
ends of the starch or related oligo- and poly-saccharide molecules. The
glucoamylase of the invention may also be used in immobilized form. This
is suitable and often used for producing speciality syrups, such as
maltose syrups as well as in the raffinate stream of oligosaccharides in
connection with the production of fructose syrups, e.g., high fructose
syrup (HFS).

[0196] The glucoamylase of the present invention can also be used for
producing various beverages, such as, but not limited to, the beverage of
tomato, potato, Chinese potato, sweet potato, and/or pumpkin.

[0198] The glucoamylases of the invention can be used in a brewing
industry. The glucoamylases of the invention is added in effective
amounts which can be easily determined by the skilled person in the art.

Production of a Liquefaction, Saccharification and/or Fermentation
Product

[0199] In this aspect the present invention relates to a process for
producing a liquefaction, saccharification and/or fermentation product
from starch-containing material, comprising the step of: treating
starch-containing material with a polypeptide of the present invention.

[0200] Suitable starch-containing starting materials are listed in the
"Starch-containing materials"-section below. Contemplated enzymes are
listed in the "Enzymes"--section below. Preferably the process of present
invention comprises treating starch-containing material with a
polypeptide of the present invention alone or together with an
alpha-amylase.

[0201] The liquefaction and/or saccharification product of the present
invention are dextrin, or low molecular sugars, for example DP1-3. In the
liquefaction process the conversion of starch into glucose, dextrin
and/or low molecular weight sugars is enhanced by the addition of a
glucoamylase of the present invention. The fermentation product, such as
ethanol, may optionally be recovered after fermentation, e.g., by
distillation. The fermentation is preferably carried out in the presence
of yeast, preferably a strain of Saccharomyces. Suitable fermenting
organisms are listed in the "Fermenting Organisms"-section below.

[0202] In this aspect the present invention relates to a process for
producing a fermentation product, especially ethanol, from
starch-containing material, which process includes a liquefaction step
and sequentially or simultaneously performed saccharification and
fermentation steps.

[0203] The invention relates to a process for producing a fermentation
product from starch-containing material comprising the steps of:

[0205] (b) saccharifying the liquefied material obtained in step (a) using
a glucoamylase; and

[0206] (c) fermenting the saccharified material using a fermenting
organism.

[0207] Preferably step (a) includes also using the glucoamylase of the
invention. In one embodiment the glucoamylase of the invention is also
present/added in step (b).

[0208] Preferably, the step of treating the starch containing material
with a polypeptide of the present invention in a liquefaction process is
performed in the presence of an alpha amylase and is carried out at
temperatures between 40° C. and 100° C., more preferably
between 80° C. and 90° C., e.g., 85° C., and at a pH
between 2.0 and 7.0, more preferably between pH 4.0 and 6.0, even more
preferably between pH 4.5 and pH 5.0, such as e.g., pH 4.8.

[0209] The fermentation product, such as especially ethanol, may
optionally be recovered after fermentation, e.g., by distillation.
Suitable starch-containing starting materials are listed in the section
"Starch-containing materials"-section below. Contemplated enzymes are
listed in the "Enzymes"-section below. The liquefaction is preferably
carried out in the presence of an alpha-amylase. The fermentation is
preferably carried out in the presence of yeast, preferably a strain of
Saccharomyces. Suitable fermenting organisms are listed in the
"Fermenting Organisms"-section below. In preferred embodiments step (b)
and (c) are carried out sequentially or simultaneously (i.e., as SSF
process).

[0210] In a particular embodiment, the process of the invention further
comprises, prior to the step (a), the steps of:

[0211] x) reducing the particle size of the starch-containing material,
preferably by milling; and

[0213] The aqueous slurry may contain from 10-40 wt. %, preferably 25-35
wt. % starch-containing material. The slurry is heated to above the
gelatinization temperature and alpha-amylase, preferably bacterial and/or
acid fungal alpha-amylase, may be added to initiate liquefaction
(thinning). The slurry may in an embodiment be jet-cooked to further
gelatinize the slurry before being subjected to an alpha-amylase in step
(a) of the invention.

[0214] More specifically liquefaction may be carried out as a three-step
hot slurry process. The slurry is heated to between 60-95° C.,
preferably 80-85° C., and alpha-amylase is added to initiate
liquefaction (thinning). Then the slurry may be jet-cooked at a
temperature between 95-140° C., preferably 105-125° C., for
1-15 minutes, preferably for 3-10 minute, especially around 5 minutes.
The slurry is cooled to 60-95° C. and more alpha-amylase is added
to finalize hydrolysis (secondary liquefaction). The liquefaction process
is usually carried out at pH 4.5-6.5, in particular at a pH between 5 and
6. Milled and liquefied whole grains are known as mash.

[0215] The saccharification in step (b) may be carried out using
conditions well know in the art. For instance, a full saccharification
process may last up to from about 24 to about 72 hours, however, it is
common only to do a pre-saccharification of typically 40-90 minutes at a
temperature between 30-65° C., typically about 60° C.,
followed by complete saccharification during fermentation in a
simultaneous saccharification and fermentation process (SSF process).
Saccharification is typically carried out at temperatures from
30-65° C., typically around 60° C., and at a pH between 4
and 5, normally at about pH 4.5.

[0216] The most widely used process in fermentation product, especially
ethanol, production is the simultaneous saccharification and fermentation
(SSF) process, in which there is no holding stage for the
saccharification, meaning that fermenting organism, such as yeast, and
enzyme(s) may be added together. SSF may typically be carried out at a
temperature between 25° C. and 40° C., such as between
29° C. and 35° C., such as between 30° C. and
34° C., such as around 32° C. According to the invention
the temperature may be adjusted up or down during fermentation.

[0218] In this aspect the invention relates to processes for producing a
fermentation product from starch-containing material without
gelatinization of the starch-containing material (i.e., uncooked
starch-containing material). According to the invention the desired
fermentation product, such as ethanol, can be produced without liquefying
the aqueous slurry containing the starch-containing material. In one
embodiment a process of the invention includes saccharifying (milled)
starch-containing material, e.g., granular starch, below the
gelatinization temperature in the presence of an alpha amylase to produce
sugars that can be fermented into the desired fermentation product by a
suitable fermenting organism. In another embodiment a glucoamylase of the
invention and an alpha amylase are used during saccharification and
fermentation. In one aspect the invention relates to a process for
producing a fermentation product from starch-containing material
comprising:

[0219] (a) saccharifying starch-containing material with a mature
glucoamylase according to the invention, preferably having the sequence
shown as amino acids 22 to 616 in SEQ ID NO: 2, at a temperature below
the initial gelatinization temperature of said starch-containing
material,

[0220] (b) fermenting using a fermenting organism.

[0221] Steps (a) and (b) of the process of the invention may be carried
out sequentially or simultaneously. In an embodiment, a slurry comprising
water and starch-containing material, is prepared before step (a).

[0222] In a preferred embodiment step (a) includes addition of an alpha
amylase.

[0223] The fermentation process may be carried out for a period of 1 to
250 hours, preferably is from 25 to 190 hours, more preferably from 30 to
180 hours, more preferably from 40 to 170 hours, even more preferably
from 50 to 160 hours, yet more preferably from 60 to 150 hours, even yet
more preferably from 70 to 140 hours, and most preferably from 80 to 130
hours.

[0224] The term "initial gelatinization temperature" means the lowest
temperature at which gelatinization of the starch commences. Starch
heated in water begins to gelatinize between 50° C. and 75°
C.; the exact temperature of gelatinization depends on the specific
starch, and can readily be determined by the skilled artisan. Thus, the
initial gelatinization temperature may vary according to the plant
species, to the particular variety of the plant species as well as with
the growth conditions. In the context of this invention the initial
gelatinization temperature of a given starch-containing material is the
temperature at which birefringence is lost in 5% of the starch granules
using the method described by Gorinstein and Lii, 1992, Starch/Starke
44(12): 461-466.

[0225] Before step (a) a slurry of starch-containing material, such as
granular starch, having 10-55 wt. % dry solids, preferably 25-40 wt. %
dry solids, more preferably 30-35 wt. % dry solids of starch-containing
material may be prepared. The slurry may include water and/or process
waters, such as stillage (backset), scrubber water, evaporator condensate
or distillate, side stripper water from distillation, or other
fermentation product plant process water. Because the process of the
invention is carried out below the gelatinization temperature and thus no
significant viscosity increase takes place, high levels of stillage may
be used if desired. In an embodiment the aqueous slurry contains from
about 1 to about 70 vol. % stillage, preferably 15-60% vol. % stillage,
especially from about 30 to 50 vol. % stillage.

[0226] The starch-containing material may be prepared by reducing the
particle size, preferably by dry or wet milling, to 0.05 to 3.0 mm,
preferably 0.1-0.5 mm. After being subjected to a process of the
invention at least 85%, at least 86%, at least 87%, at least 88%, at
least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or
preferably at least 99% of the dry solids of the starch-containing
material is converted into a soluble starch hydrolysate.

[0227] The process of the invention is conducted at a temperature below
the initial gelatinization temperature. Preferably the temperature at
which step (a) is carried out is between 30-75° C., preferably
between 45-60° C.

[0228] In a preferred embodiment step (a) and step (b) are carried out as
a sequential or simultaneous saccharification and fermentation process.
In such preferred embodiment the process is typically carried at a
temperature between 25° C. and 40° C., such as between
29° C. and 35° C., such as between 30° C. and
34° C., such as around 32° C. According to the invention
the temperature may be adjusted up or down during fermentation.

[0229] In an embodiment simultaneous saccharification and fermentation is
carried out so that the sugar level, such as glucose level, is kept at a
low level such as below 6 wt. %, preferably below about 3 wt. %,
preferably below about 2 wt. %, more preferred below about 1 wt. %., even
more preferred below about 0.5 wt. %, or even more preferred 0.25 wt. %,
such as below about 0.1 wt. %. Such low levels of sugar can be
accomplished by simply employing adjusted quantities of enzyme and
fermenting organism. A skilled person in the art can easily determine
which quantities of enzyme and fermenting organism to use. The employed
quantities of enzyme and fermenting organism may also be selected to
maintain low concentrations of maltose in the fermentation broth. For
instance, the maltose level may be kept below about 0.5 wt. % or below
about 0.2 wt. %.

[0230] The process may be carried out at a pH in the range between 3 and
7, preferably from pH 3.5 to 6, or more preferably from pH 4 to 5.

Starch-Containing Materials

[0231] Any suitable starch-containing starting material, including
granular starch, may be used according to the present invention. The
starting material is generally selected based on the desired fermentation
product. Examples of starch-containing starting materials, suitable for
use in a process of present invention, include tubers, roots, stems,
whole grains, corns, cobs, wheat, barley, rye, milo, sago, cassava,
tapioca, sorghum, rice peas, beans, or sweet potatoes, or mixtures
thereof, or cereals, sugar-containing raw materials, such as molasses,
fruit materials, sugar cane or sugar beet, potatoes, and
cellulose-containing materials, such as wood or plant residues, or
mixtures thereof. Contemplated are both waxy and non-waxy types of corn
and barley.

Fermenting Organisms

[0232] "Fermenting organism" refers to any organism, including bacterial
and fungal organisms, suitable for use in a fermentation process and
capable of producing desired a fermentation product. Especially suitable
fermenting organisms are able to ferment, i.e., convert, sugars, such as
glucose or maltose, directly or indirectly into the desired fermentation
product. Examples of fermenting organisms include fungal organisms, such
as yeast. Preferred yeast includes strains of Saccharomyces spp., in
particular, Saccharomyces cerevisiae. Commercially available yeast
include, e.g., Red StarTm/Lesaffre Ethanol Red (available from Red
Star/Lesaffre, USA) FALI (available from Fleischmann's Yeast, a division
of Burns Philp Food Inc., USA), SUPERSTART (available from Alltech), GERT
STRAND (available from Gert Strand AB, Sweden) and FERMIOL (available
from DSM Specialties).

Enzymes

Glucoamylase

[0233] The glucoamylase is preferably a glucoamylase of the invention.
However, as mentioned above a glucoamylase of the invention may also be
combined with other glucoamylases.

[0235] The alpha-amylase may according to the invention be of any origin.
Preferred are alpha-amylases of fungal or bacterial origin.

[0236] In a preferred aspect the alpha-amylase is an acid alpha-amylase,
e.g., fungal acid alpha-amylase or bacterial acid alpha-amylase. The term
"acid alpha-amylase" means an alpha-amylase (E.C. 3.2.1.1) which added in
an effective amount has activity optimum at a pH in the range of 3 to 7,
preferably from 3.5 to 6, or more preferably from 4-5.

Bacterial Alpha-Amylases

[0237] According to the invention a bacterial alpha-amylase may preferably
be derived from the genus Bacillus.

[0238] In a preferred aspect the Bacillus alpha-amylase is derived from a
strain of B. licheniformis, B. amyloliquefaciens, B. subtilis or B.
stearothermophilus, but may also be derived from other Bacillus sp.
Specific examples of contemplated alpha-amylases include the Bacillus
licheniformis alpha-amylase (BLA) shown in SEQ ID NO: 4 in WO 99/19467,
the Bacillus amyloliquefaciens alpha-amylase (BAN) shown in SEQ ID NO: 5
in WO 99/19467, and the Bacillus stearothermophilus alpha-amylase (BSG)
shown in SEQ ID NO: 3 in WO 99/19467. In an embodiment of the invention
the alpha-amylase is an enzyme having a degree of identity of at least
60%, preferably at least 70%, more preferred at least 80%, even more
preferred at least 90%, such as at least 95%, at least 96%, at least 97%,
at least 98% or at least 99% identity to any of the sequences shown as
SEQ ID NOS: 1, 2, 3, 4, or 5, respectively, in WO 99/19467.

[0239] The Bacillus alpha-amylase may also be a variant and/or hybrid,
especially one described in any of WO 96/23873, WO 96/23874, WO 97/41213,
WO 99/19467, WO 00/60059, and WO 02/10355 (all documents hereby
incorporated by reference). Specifically contemplated alpha-amylase
variants are disclosed in U.S. Pat. Nos. 6,093,562, 6,297,038 or U.S.
Pat. No. 6,187,576 (hereby incorporated by reference) and include
Bacillus stearothermophilus alpha-amylase (BSG alpha-amylase) variants
having a deletion of one or two amino acid in position 179 to 182,
preferably a double deletion disclosed in WO 96/23873-see e.g., page 20,
lines 1-10 (hereby incorporated by reference), preferably corresponding
to delta(181-182) compared to the wild-type BSG alpha-amylase amino acid
sequence set forth in SEQ ID NO: 3 disclosed in WO 99/19467 or deletion
of amino acids 179 and 180 using SEQ ID NO: 3 in WO 99/19467 for
numbering (which reference is hereby incorporated by reference). Even
more preferred are Bacillus alpha-amylases, especially Bacillus
stearothermophilus alpha-amylase, which have a double deletion
corresponding to delta(181-182) and further comprise a N193F substitution
(also denoted 1181*+G182*+N193F) compared to the wild-type BSG
alpha-amylase amino acid sequence set forth in SEQ ID NO: 3 disclosed in
WO 99/19467.

[0240] The alpha-amylase may also be a maltogenic alpha-amylase. A
"maltogenic alpha-amylase" (glucan 1,4-alpha-maltohydrolase, E.C.
3.2.1.133) is able to hydrolyze amylose and amylopectin to maltose in the
alpha-configuration. A maltogenic alpha-amylase from Bacillus
stearothermophilus strain NCIB 11837 is commercially available from
Novozymes NS, Denmark. The maltogenic alpha-amylase is described in U.S.
Pat. Nos. 4,598,048, 4,604,355 and 6,162,628, which are hereby
incorporated by reference.

Bacterial Hybrid Alpha-Amylases

[0241] A hybrid alpha-amylase specifically contemplated comprises 445
C-terminal amino acid residues of the Bacillus licheniformis
alpha-amylase (shown as SEQ ID NO: 4 in WO 99/19467) and the 37
N-terminal amino acid residues of the alpha-amylase derived from Bacillus
amyloliquefaciens (shown as SEQ ID NO: 3 in WO 99/194676), with one or
more, especially all, of the following substitutions:
G48A+T49I+G107A+H156Y+A181T+N190F+1201F+A209V+Q264S (using the Bacillus
licheniformis numbering). Also preferred are variants having one or more
of the following mutations (or corresponding mutations in other Bacillus
alpha-amylase backbones): H154Y, A181T, N190F, A209V and Q264S and/or
deletion of two residues between positions 176 and 179, preferably
deletion of E178 and G179 (using the SEQ ID NO: 5 numbering of WO
99/19467).

Fungal Alpha-Amylases

[0242] Fungal acid alpha-amylases include acid alpha-amylases derived from
a strain of the genus Aspergillus, such as Aspergillus oryzae,
Aspergillus niger, or Aspergillus kawachii alpha-amylases. A preferred
acid fungal alpha-amylase is a Fungamyl-like alpha-amylase which is
preferably derived from a strain of Aspergillus oryzae. In the present
disclosure, the term "Fungamyl-like alpha-amylase" indicates an
alpha-amylase which exhibits a high identity, i.e. more than 70%, more
than 75%, more than 80%, more than 85% more than 90%, more than 95%, more
than 96%, more than 97%, more than 98%, more than 99% or even 100%
identity to the mature part of the amino acid sequence shown in SEQ ID
NO: 10 in WO 96/23874.

[0243] Another preferred acid alpha-amylase is derived from a strain
Aspergillus niger. In a preferred aspect the acid fungal alpha-amylase is
the one from A. niger disclosed as "AMYA_ASPNG" in the Swiss-prot/TeEMBL
database under the primary accession no. P56271 and described in more
detail in WO 89/01969 (Example 3). The acid Aspergillus niger acid
alpha-amylase is also shown as SEQ ID NO: 1 in WO 2004/080923 (Novozymes)
which is hereby incorporated by reference. Also variants of said acid
fungal amylase having at least 70% identity, such as at least 80% or even
at least 90% identity, such as at least 95%, at least 96%, at least 97%,
at least 98%, or at least 99% identity to SEQ ID NO: 1 in WO 2004/080923
are contemplated.

[0244] In a preferred aspect the alpha-amylase is derived from Aspergillus
kawachii and disclosed by Kaneko et al., 1996, J. Ferment. Bioeng. 81:
292-298, "Molecular-cloning and determination of the nucleotide-sequence
of a gene encoding an acid-stable alpha-amylase from Aspergillus
kawachii"; and further as EMBL:#AB008370.

[0245] The fungal acid alpha-amylase may also be a wild-type enzyme
comprising a carbohydrate-binding module (CBM) and an alpha-amylase
catalytic domain (i.e., a none-hybrid), or a variant thereof. In an
embodiment the wild-type acid alpha-amylase is derived from a strain of
Aspergillus kawachii.

[0246] An acid alpha-amylases may according to the invention be added in
an amount of 0.01 to 10 AFAU/g DS, preferably 0.01 to 5 AFAU/g DS,
especially 0.02 to 2 AFAU/g DS.

Fungal Hybrid Alpha-Amylases

[0247] In a preferred aspect the fungal acid alpha-amylase is a hybrid
alpha-amylase. Preferred examples of fungal hybrid alpha-amylases include
the ones disclosed in WO 2005/003311 or U.S. Patent Publication no.
2005/0054071 (Novozymes) or US patent application No. 2006/0148054
(Novozymes) which is hereby incorporated by reference. A hybrid
alpha-amylase may comprise an alpha-amylase catalytic domain (CD) and a
carbohydrate-binding domain/module (CBM) and optional a linker.

[0249] Other specific examples of contemplated hybrid alpha-amylases
include, but not limited to those disclosed in U.S. Patent Publication
no. 2005/0054071, including those disclosed in Table 3 on page 15, such
as Aspergillus niger alpha-amylase with Aspergillus kawachii linker and
starch binding domain.

[0256] Glucoamylase (AMG), EC 3.2.1.3
(exo-alpha-1,4-glucan-glucohydrolase), hydrolyzes maltose to form
alpha-D-glucose. After incubation, the reaction is stopped with NaOH.

[0257] Steps 2 and 3 result in an endpoint reaction:

[0258] Glucose is phosphorylated by ATP, in a reaction catalyzed by
hexokinase. The glucose-6-phosphate formed is oxidized to
6-phosphogluconate by glucose-6-phosphate dehydrogenase. In this same
reaction, an equimolar amount of NAD+ is reduced to NADH with a resulting
increase in absorbance at 340 nm. An autoanalyzer system such as Konelab
30 Analyzer (Thermo Fisher Scientific) may be used.

[0259] When used according to the present invention the activity of any
acid alpha-amylase may be measured in AFAU (Acid Fungal Alpha-amylase
Units). Alternatively activity of acid alpha-amylase may be measured in
KNU-s (Kilo Novozymes Units (Termamyl SC)).

[0261] Acid alpha-amylase, an endo-alpha-amylase
(1,4-alpha-D-glucan-glucanohydrolase, E.C. 3.2.1.1) hydrolyzes
alpha-1,4-glucosidic bonds in the inner regions of the starch molecule to
form dextrins and oligosaccharides with different chain lengths. The
intensity of color formed with iodine is directly proportional to the
concentration of starch. Amylase activity is determined using reverse
colorimetry as a reduction in the concentration of starch under the
specified analytical conditions.

[0265] E. coli transformation for DNA sequencing was carried out by
electroporation (BIO-RAD Gene Pulser) or chemically. DNA Plasmids were
prepared by alkaline method (Molecular Cloning, Cold Spring Harbor) or
with the Qiagen® Plasmid Kit. DNA fragments were recovered from
agarose gel by the Qiagen gel extraction Kit. PCR was performed using a
PTC-200 DNA Engine. The ABI PRISM® 310 Genetic Analyzer was used for
determination of all DNA sequences.

[0287] The Penicillium oxalicum glucoamylase gene was re-cloned from the
plasmid AMG 1 into an Aspergillus expression vector by PCR using two
cloning primer F and primer R shown below, which were designed based on
the known sequence and added tags for direct cloning by IN-FUSION®
strategy.

[0288] A PCR reaction was performed with plasmid AMG 1 in order to amplify
the full-length gene. The PCR reaction was composed of 40 pg of the
plasmid AMG 1 DNA, 1 μl of each primer (100 μM); 12.5 μl of
2× Extensor Hi-Fidelity master mix (Extensor Hi-Fidelity Master
Mix, ABgene, United Kingdom), and 9.5 μl of PCR-grade water. The PCR
reaction was performed using a DYAD PCR machine (Bio-Rad Laboratories,
Inc., Hercules, Calif., USA) programmed for 2 minutes at 94° C.
followed by a 25 cycles of 94° C. for 15 seconds, 50° C.
for 30 seconds, and 72° C. for 1 minute; and then 10 minutes at
72° C.

[0289] The reaction products were isolated by 1.0% agarose gel
electrophoresis using 1×TAE buffer where an approximately 1.9 kb
PCR product band was excised from the gel and purified using a GFX®
PCR DNA and Gel Band Purification Kit (GE Healthcare, United Kingdom)
according to manufacturer's instructions. DNA corresponding to the
Penicillium oxalicum glucoamylase gene was cloned into an Aspergillus
expression vector linearized with BamHl and HindIII, using an
IN-FUSION® Dry-Down PCR Cloning Kit (BD Biosciences, Palo Alto,
Calif., USA) according to the manufacturer's instructions. The linearized
vector construction is as described in WO 2005/042735 A1.

[0290] A 2 μl volume of the ligation mixture was used to transform 25
μl of Fusion Blue E. coli cells (included in the IN-FUSION®
Dry-Down PCR Cloning Kit). After a heat shock at 42° C. for 45
sec, and chilling on ice, 250 μl of SOC medium was added, and the
cells were incubated at 37° C. at 225 rpm for 90 min before being
plated out on LB agar plates containing 50 μg of ampicillin per ml,
and cultivated overnight at 37° C. Selected colonies were
inoculated in 3 ml of LB medium supplemented with 50 μg of ampicillin
per ml and incubated at 37° C. at 225 rpm overnight. Plasmid DNA
from the selected colonies was purified using Mini JETSTAR (Genomed,
Germany) according to the manufacturer's instructions. Penicillium
oxalicum glucoamylase gene sequence was verified by Sanger sequencing
before heterologous expression. One of the plasmids was selected for
further expression, and was named XYZ XYZ1471-4.

[0291] Protoplasts of Aspergillus niger MBin118 were prepared as described
in WO 95/02043. One hundred μl of protoplast suspension were mixed
with 2.5 μg of the XYZ1471-4 plasmid and 250 microliters of 60% PEG
4000 (Applichem) (polyethylene glycol, molecular weight 4,000), 10 mM
CaCl2, and 10 mM Tris-HCl pH 7.5 were added and gently mixed. The
mixture was incubated at 37° C. for 30 minutes and the protoplasts
were mixed with 6% low melting agarose (Biowhittaker Molecular
Applications) in COVE sucrose (Cove, 1996, Biochim. Biophys. Acta
133:51-56) (1 M) plates supplemented with 10 mM acetamid and 15 mM CsCl
and added as a top layer on COVE sucrose (1 M) plates supplemented with
10 mM acetamid and 15 mM CsCl for transformants selection (4 ml topagar
per plate). After incubation for 5 days at 37° C. spores of
sixteen transformants were picked up and seed on 750 μl YP-2% Maltose
medium in 96 deepwell MT plates. After 5 days of stationary cultivation
at 30° C., 10 μl of the culture-broth from each well was
analyzed on a SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis) gel, Griton XT Precast gel (BioRad, CA, USA) in order to
identify the best transformants based on the ability to produce large
amount of glucoamylase. A selected transformant was identified on the
original transformation plate and was preserved as spores in a 20%
glycerol stock and stored frozen (-80° C.).

[0292] Cultivation.

[0293] The selected transformant was inoculated in 100 ml of MLC media and
cultivated at 30° C. for 2 days in 500 ml shake flasks on a rotary
shaker. 3 ml of the culture broth was inoculated to 100 ml of M410 medium
and cultivated at 30° C. for 3 days. The culture broth was
centrifugated and the supernatant was filtrated using 0.2 μm membrane
filters.

[0294] Alpha-Cyclodextrin Affinity Gel.

[0295] Ten grams of Epoxy-activated Sepharose 6B (GE Healthcare, Chalfont
St. Giles, U.K) powder was suspended in and washed with distilled water
on a sintered glass filter. The gel was suspended in coupling solution
(100 ml of 12.5 mg/ml alpha-cyclodextrin, 0.5 M NaOH) and incubated at
room temperature for one day with gentle shaking. The gel was washed with
distilled water on a sintered glass filter, suspended in 100 ml of 1 M
ethanolamine, pH 10, and incubated at 50° C. for 4 hours for
blocking. The gel was then washed several times using 50 mM Tris-HCl, pH
8 and 50 mM NaOAc, pH 4.0 alternatively. The gel was finally packed in a
35-40 ml column using equilibration buffer (50 mM NaOAc, 150 mM NaCl, pH
4.5).

[0296] Purification of Glucoamylase from Culture Broth.

[0297] Culture broth from fermentation of A. niger MBin118 harboring the
glucoamylase gene was filtrated through a 0.22 μm PES filter, and
applied on a alpha-cyclodextrin affinity gel column previously
equilibrated in 50 mM NaOAc, 150 mM NaCl, pH 4.5 buffer. Unbound material
was washed off the column with equilibration buffer and the glucoamylase
was eluted using the same buffer containing 10 mM beta-cyclodextrin over
3 column volumes. The glucoamylase activity of the eluent was checked to
see, if the glucoamylase had bound to the alpha-cyclodextrin affinity
gel. The purified glucoamylase sample was then dialyzed against 20 mM
NaOAc, pH 5.0. The purity was finally checked by SDS-PAGE, and only a
single band was found.

Example 3

Construction and Expression of a Site-Directed Variant of Penicillium
oxalicum Glucoamylase

[0298] Two PCR reactions were performed with plasmid XYZ1471-4, described
in Example 2, using primers K79V F and K79V R shown below, which were
desined to substitute lysine (K) at position 79 from the mature seequence
to valine (V) and primers F-NP003940 and R-NP003940 shown below, which
were designed based on the known sequence and added tags for direct
cloning by IN-FUSION® strategy.

[0300] DNA fragments were recovered from agarose gel by the Qiagen gel
extraction Kit according to the manufacturer's instruction. The resulting
purified two fragments were cloned into an Aspergillus expression vector
linearized with BamHl and HindIII, using an IN-FUSION® Dry-Down PCR
Cloning Kit (BD Biosciences, Palo Alto, Calif., USA) according to the
manufacturer's instructions. The linearized vector construction is as
described in WO 2005/042735 A1.

[0301] The ligation mixture was used to transform E. coli DH5a cells
(TOYOBO). Selected colonies were inoculated in 3 ml of LB medium
supplemented with 50 μg of ampicillin per ml and incubated at
37° C. at 225 rpm overnight. Plasmid DNA from the selected
colonies was purified using Qiagen plasmid mini kit (Qiagen) according to
the manufacturer's instructions. The sequence of Penicillium oxalicum
glucoamylase site-directed variant gene sequence was verified before
heterologous expression and one of the plasmids was selected for further
expression, and was named pPoPE001.

[0302] Protoplasts of Aspergillus niger MBin118 were prepared as described
in WO 95/02043. One hundred μl of protoplast suspension were mixed
with 2.5 μg of the pPoPE001 plasmid and 250 microliters of 60% PEG
4000 (Applichem) (polyethylene glycol, molecular weight 4,000), 10 mM
CaCl2, and 10 mM Tris-HCl pH 7.5 were added and gently mixed. The
mixture was incubated at 37° C. for 30 minutes and the protoplasts
were mixed with 1% agarose L (Nippon Gene) in COVE sucrose (Cove, 1996,
Biochim. Biophys. Acta 133:51-56) supplemented with 10 mM acetamid and 15
mM CsCl and added as a top layer on COVE sucrose plates supplemented with
10 mM acetamid and 15 mM CsCl for transformants selection (4 ml topagar
per plate). After incubation for 5 days at 37° C. spores of
sixteen transformants were picked up and seed on 750 μl YP-2% Maltose
medium in 96 deepwell MT plates. After 5 days of stationary cultivation
at 30° C., 10 μl of the culture-broth from each well was
analyzed on a SDS-PAGE gel in order to identify the best transformants
based on the ability to produce large amount of the glucoamylase.

Example 4

Purification of Site-Directed Po AMG Variant PE001

[0303] The selected transformant of the variant and the strain expressing
the wilid type Penicillium oxalicum glucoamylase described in Example 1
was cultivated in 100 ml of YP-2% maltose medium and the culture was
filtrated through a 0.22 μm PES filter, and applied on a
alpha-cyclodextrin affinity gel column previously equilibrated in 50 mM
NaOAc, 150 mM NaCl, pH 4.5 buffer. Unbound material was washed off the
column with equilibration buffer and the glucoamylase was eluted using
the same buffer containing 10 mM beta-cyclodextrin over 3 column volumes.

[0304] The glucoamylase activity of the eluent was checked to see, if the
glucoamylase had bound to the alpha-cyclodextrin affinity gel. The
purified glucoamylase samples were then dialyzed against 20 mM NaOAc, pH
5.0.

Example 5

Characterization of PE001

Protease Stability

[0305] 40 μl enzyme solutions (1 mg/ml) in 50 mM sodium acetate buffer,
pH 4.5, was mixed with 1/10 volume of 1 mg/ml protease solutions such as
aspergillopepsinl described in Biochem J. 147(1):45-53 (1975). or the
commercially availble product from Sigma and aorsin described in
Ichishima, 2003, Biochemical Journal 371(2): 541 and incubated at 4 or
32° C. overnight. As a control experiment, H2O was added to
the sample instead of proteases. The samples were loaded on SDS-PAGE to
see if the glucoamylases are cleaved by proteases.

[0306] In SDS-PAGE, PE001 only showed one band corresponding to the intact
molecule, while the wild type glucoamylase was degraded by proteases and
showed a band at lower molecular size at 60 kDa.

[0312] The Penicillium oxalicum AMG (PoAMG) variant, PE001, showing
reduced sensitivity to protease degradadtion, was tested in both whole
corn liquefaction and starch saccharification (shown in next section).
For the whole corn liquefactions, the PE001 enzyme was added in different
doses with a low pH amylase variant, Alpha amylase 1407. In some
liquefactions, the PE001 variant was tested with both the low pH amylase
Alpha amylase 1407 and the thermostable protease Protease 196. In all
experiments, the liquefactions were done using the automated system
called the "Lab-O-Mat". This instrument controls the temperature and
provides constant mixing. The other experimental conditions were: pH was
4.8 (for the liquefacts containing the AA1407 low pH amylase) or 5.8 (for
the Alpha Amylase A control), 32% dry solids, 85° C., 2 hours
total time. The enzyme dosing schemes are shown in Table 4. The liquefied
mashes were saccharified and fermented using a composition comprising
Talaromyces emersonii AMG as the main activity and Trametes cingulata AMG
and Hybrid AA as side activities (80%/19%/1%)(at a dose of 0.5 AGU/gram
dry solids for 54 hours at 32° C.

Characterization of Protease Stability of Variants Having Alternative
Substitutions at Position 79

[0314] Variants comprising substitutions at position 79 of SEQ ID NO: 2,
K79A, K79G, K791, K79L, K79S, K79T, were constructed as described in
Example 3 using the appropriate primers.

[0315] Each of the variants were cultivated and purified as described in
Example 4 and the protease stability was tested as described below.

Protease Stability

[0316] 20 μl enzyme solution (1 mg/ml) in 50 mM sodium acetate buffer,
pH 4.5, was mixed with 1/10 volume of 1 mg/ml protease solutions such as
aorsin described in Ichishima, 2003, Biochemical Journal 371: 541) and
incubated at -20 or 37° C. overnight. As a control experiment,
H2O was added to the sample instead of proteases. The samples were
loaded on SDS-PAGE to see if the glucoamylase variants are cleaved by
proteases.

[0317] On SDS-PAGE, all variants only showed one band corresponding to the
intact molecule (70 kDa), while the wild type glucoamylase was degraded
and showed a band at lower molecular size at 60 kDa.

[0318] The invention described and claimed herein is not to be limited in
scope by the specific aspects herein disclosed, since these aspects are
intended as illustrations of several aspects of the invention. Any
equivalent aspects are intended to be within the scope of this invention.
Indeed, various modifications of the invention in addition to those shown
and described herein will become apparent to those skilled in the art
from the foregoing description. Such modifications are also intended to
fall within the scope of the appended claims. In the case of conflict,
the present disclosure including definitions will control.

[0319] The present invention is further described by the following
numbered paragraphs.

[1] A glucoamylase variant, comprising a substitution at least at a
position corresponding to positions 79 of the mature polypeptide of SEQ
ID NO: 2, wherein the variant has glucoamylase activity. [2] The variant
of paragraph 1, selected from the group consisting of:

[0320] a) a polypeptide having at least 65% sequence identity to the
mature polypeptide of SEQ ID NO: 2;

[0321] b) a polypeptide encoded by a polynucleotide that hybridizes under
low stringency conditions with (i) the mature polypeptide coding sequence
of SEQ ID NO: 1, or (ii) the full-length complement of (i);

[0322] c) a polypeptide encoded by a polynucleotide having at least 65%
identity to the mature polypeptide coding sequence of SEQ ID NO: 1; and

[0323] d) a fragment of the mature polypeptide of SEQ ID NO: 2, which has
glucoamylase activity.

[3] The variant of paragraph 1 or 2, wherein the variant has at least at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at
least 99% sequence identity to the mature polypeptide of SEQ ID NO: 2.
[4] The variant of paragraph 1 or 2, wherein the variant is encoded by a
polynucleotide that hybridizes under low stringency conditions, medium
stringency conditions, medium-high stringency conditions, high stringency
conditions, or very high stringency conditions with (i) the mature
polypeptide coding sequence of SEQ ID NO: 1, or (ii) the full-length
complement of (i). [5] The variant of paragraph 1 or 2, wherein the
variant is encoded by a polynucleotide having at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99% sequence identity to
the mature polypeptide coding sequence of SEQ ID NO: 1. [6] The variant
of any of paragraphs 1-5, which is a variant of a parent glucoamylase
selected from the group consisting of:

[0324] a) a polypeptide having at least 65% sequence identity to the
mature polypeptide of SEQ ID NO: 2;

[0325] b) a polypeptide encoded by a polynucleotide that hybridizes under
low stringency conditions with (i) the mature polypeptide coding sequence
of SEQ ID NO: 1, or (ii) the full-length complement of (i);

[0326] c) a polypeptide encoded by a polynucleotide having at least 65%
identity to the mature polypeptide coding sequence of SEQ ID NO: 1; and

[0327] d) a fragment of the mature polypeptide of SEQ ID NO: 2, which has
glucoamylase activity.

[7] The variant of paragraph 6, wherein the parent glucoamylase has at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99% or 100% sequence identity to the mature polypeptide of SEQ ID
NO: 2. [8] The variant of paragraph 6 or 7, wherein the parent
glucoamylase is encoded by a polynucleotide that hybridizes under low
stringency conditions, medium stringency conditions, medium-high
stringency conditions, high stringency conditions, or very high
stringency conditions with (i) the mature polypeptide coding sequence of
SEQ ID NO: 1 or (ii) the full-length complement of (i). [9] The variant
of any of paragraphs 6-8, wherein the parent glucoamylase is encoded by a
polynucleotide having at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at least 98%, at least 99%, or 100% sequence identity to the mature
polypeptide coding sequence of SEQ ID NO: 1. [10] The variant of any of
paragraphs 1-9, wherein the number of substitutions is 1-20, e.g., 1-10
and 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions. [11] The
variant of any of paragraphs 1-10, wherein the variant comprises a
substitution selected from K79V, K79A, K79G, K791, K79L, K79S, K79T. [12]
The variant of any of paragraphs 1-11, wherein the variant comprises
substitution K79V. [13] The variant of any of paragraphs 1-12, which has
an improved property relative to the parent, wherein the improved
property is reduced sensitivity to protease degradation. [14] A variant
glucoamylase catalytic domain comprising a substitution at least at a
position corresponding to positions 79 of the mature polypeptide of SEQ
ID NO: 2, wherein the variant has glucoamylase activity. [15] The variant
glucoamylase catalytic domain of paragraph 14 selected from the group
consisting of:

[0329] (b) a catalytic domain encoded by a polynucleotide that hybridizes
under medium stringency conditions with (i) nucleotides 88 to 1482 of SEQ
ID NO: 1 or (ii) the full-length complement of (i);

[0330] (c) a catalytic domain encoded by a polynucleotide having at least
65% sequence identity to (i) nucleotides 88 to 1482 of SEQ ID NO: 1; and

[0331] (d) a variant of amino acids 30 to 494 of SEQ ID NO: 2 comprising a
substitution, deletion, and/or insertion at one or more (e.g., several)
positions; and wherein the catalytic domain has glucoamylase activity.

[16] The polypeptide of paragraph 15, further comprising a linker and a
carbohydrate binding domain. [17] A composition comprising the
polypeptide of any of paragraphs 1-16. [18] The composition of paragraph
17, comprising an alpha-amylase and a polypeptide of any of paragraphs
1-16. [19] A use of a polypeptide of any of paragraphs 1-16 for
production of syrup and/or a fermentation product. [20] The use of
paragraph 19, wherein the starting material is gelatinized or
un-gelatinized starch-containing material. [21] A use of a polypeptide of
any of paragraphs 1-16 for brewing. [22] A process of producing a
fermentation product from starch-containing material comprising the steps
of:

[0332] (a) liquefying starch-containing material in the presence of an
alpha amylase;

[0333] (b) saccharifying the liquefied material; and

[0334] (c) fermenting with a fermenting organism; wherein step (a) and/or
step (b) is carried out using at least a glucoamylase of any of
paragraphs 1-16.

[23] A process of producing a fermentation product from starch-containing
material, comprising the steps of:

[0335] (a) saccharifying starch-containing material at a temperature below
the initial gelatinization temperature of said starch-containing
material; and

[0336] (b) fermenting with a fermenting organism, wherein step (a) is
carried out using at least a glucoamylase of any of paragraphs 1-16.

[24] An isolated polynucleotide encoding the polypeptide of any of
paragraphs 1-16. [25] A nucleic acid construct or expression vector
comprising the polynucleotide of paragraph 24 operably linked to one or
more control sequences that direct the production of the polypeptide in
an expression host. [26] A recombinant host cell comprising the
polynucleotide of paragraph 24 operably linked to one or more control
sequences that direct the production of the polypeptide. [27] A method of
producing the polypeptide of any of paragraphs 1-16, comprising:

[0337] (a) cultivating a cell, which in its wild-type form produces the
polypeptide, under conditions conducive for production of the
polypeptide; and

[0338] (b) recovering the polypeptide.

[28] A method of producing a polypeptide of any of paragraphs 1-16,
comprising:

[0339] (a) cultivating the host cell of paragraph 26 under conditions
conducive for production of the polypeptide; and